OSWG Recommended Approaches to the Nonclinical Pharmacokinetic (ADME) Characterization of Therapeutic Oligonucleotides.

This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.

Scientific rationale for a higher dose of nusinersen.

OBJECTIVE: The long-term favorable safety profile of nusinersen provides an opportunity to consider a higher dose. We report on the relationships between nusinersen cerebrospinal fluid (CSF) exposure, biomarker levels, and clinical efficacy. METHODS: The analyses used data from the CS3A and ENDEAR studies of nusinersen in participants with infantile-onset spinal muscular atrophy (SMA). Steady-state CSF trough (Ctrough ) levels, plasma phosphorylated neurofilament heavy chain (pNF-H) levels, body weight, and Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scores were selected as parameters of interest. A validated population pharmacokinetic (PK) model was applied to predict the nusinersen CSF Ctrough . PK/pharmacodynamic (PK/PD) models used nusinersen CSF Ctrough measurements, which were time-matched with CHOP INTEND scores. RESULTS: Higher nusinersen CSF exposure was associated with a greater decrease in pNF-H levels and greater efficacy, as measured by change in the CHOP INTEND score from baseline. These findings indicate a dose-response relationship between CSF nusinersen levels and treatment response. The higher dose is predicted to lead to approximately a 2.4-fold increase in nusinersen CSF levels with fewer loading doses. PK/PD modeling indicates that a higher concentration of nusinersen may predict an additional 5-point increase in CHOP INTEND score beyond that observed with 12 mg. INTERPRETATION: Our data indicate that a higher dose of nusinersen may lead to additional clinically meaningful improvement in efficacy when compared with the currently approved 12-mg dose. The efficacy, safety, and PK of a higher nusinersen dose are currently under investigation in the ongoing phase 2/3 DEVOTE study (NCT04089566).

Restoration of Nusinersen Levels Following Treatment Interruption in People With Spinal Muscular Atrophy: Simulations Based on a Population Pharmacokinetic Model.

BACKGROUND: Nusinersen is approved for the treatment of spinal muscular atrophy. The most common approved dosing regimen is four intrathecal loading doses of nusinersen 12 mg; the first three are administered at 14-day intervals followed by a fourth dose 30 days later, and then 12-mg maintenance doses are administered every 4 months thereafter. Interruption of nusinersen treatment in the maintenance dosing phase might occur for a number of clinical reasons. OBJECTIVE: The objective of this report is to describe dosing regimens that allow for the most rapid restoration of steady-state concentrations of nusinersen in the cerebrospinal fluid (CSF) following a treatment interruption during maintenance dosing. METHODS: Population pharmacokinetic models using integrated pharmacokinetic data from ten nusinersen clinical trials that included a broad range of participants with spinal muscular atrophy treated with intrathecal nusinersen were used to investigate different durations of treatment interruptions during maintenance treatment. Potential dosing regimens for re-initiation of nusinersen were evaluated, with the goal of achieving the quickest restoration of steady-state nusinersen CSF concentrations without exceeding maximal CSF exposures observed during the initial loading period. RESULTS: Our pharmacokinetic modeling indicates the following regimen will lead to optimal restoration of nusinersen CSF levels after treatment interruption: two doses of nusinersen should be administered at 14-day intervals following treatment interruptions of ≥ 8 to < 16 months since the last dose, and three doses of nusinersen at 14-day intervals for treatment interruptions of ≥ 16 to < 40 months since the last maintenance dose, with subsequent maintenance dosing every 4 months in both instances. After treatment interruptions of ≥ 40 months, the full loading regimen will rapidly restore nusinersen CSF levels. CONCLUSIONS: Prolonged treatment interruptions lead to suboptimal CSF levels of nusinersen. The optimal regimen to restore nusinersen CSF levels depends on the interval since the last maintenance dose was administered.

Nusinersen is a drug used to treat people of all ages who have spinal muscular atrophy. Nusinersen is injected with a thin needle into the lower back, a procedure known as a lumbar puncture. People initially receive three doses of nusinersen 12 mg each 14 days apart. They receive a fourth dose 1 month later, and then injections every 4 months (known as maintenance dosing). This treatment plan allows nusinersen to build up to effective levels in the fluid surrounding the spinal cord and brain. Some people may miss dose(s) or may stop nusinersen treatment at some point during maintenance dosing and then may want to continue treatment. This study used information from ten clinical trials to find out the best way to restart treatment to build up nusinersen to effective levels. People with a treatment break of ≥ 8 to < 16 months since the last dose need two doses of nusinersen at 14-day intervals before receiving maintenance dosing. People with a treatment break of ≥ 16 to < 40 months since the last dose need three doses of nusinersen at 14-day intervals before receiving maintenance dosing. If people stopped treatment for ≥ 40 months, they would need four doses before starting maintenance treatment. Results from this study showed that the number of doses that people needed before starting maintenance treatment depended on how long the treatment break was.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling.

PURPOSE: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. METHODS: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. RESULTS: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). CONCLUSIONS: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

Evaluation of Floxuridine Oligonucleotide Conjugates Carrying Potential Enhancers of Cellular Uptake.

Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates provide the highest antiproliferative activity without having the highest internalization results. On the contrary, cholesterol oligomers that were the best-internalized oligomers had poor antiproliferative activity, even worse than the unmodified floxuridine oligomer. Especially relevant is the effect induced by palmitic and folic acid derivatives generating the most active drugs. These results are of special interest for delivering other therapeutic oligonucleotides.

Safety, tolerability, and pharmacokinetics of casimersen in patients with Duchenne muscular dystrophy amenable to exon 45 skipping: A randomized, double-blind, placebo-controlled, dose-titration trial.

INTRODUCTION/AIMS: Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene resulting in the absence of dystrophin. Casimersen is a phosphorodiamidate morpholino oligomer designed to bypass frameshift DMD mutations and produce internally truncated, yet functional, dystrophin protein in patients amenable to exon 45 skipping. Our primary study objective was to evaluate safety and tolerability of casimersen; the secondary objective was to characterize the plasma pharmacokinetics. METHODS: This multicenter, phase 1/2 trial enrolled 12 participants (aged 7-21 years, who had limited ambulation or were nonambulatory) and comprised a 12-week, double-blind dose titration, then an open-label extension for up to 132 weeks. During dose titration, participants were randomized 2:1 to weekly casimersen infusions at escalating doses of 4, 10, 20, and 30 mg/kg (≥2 weeks per dose), or placebo. RESULTS: Participants received casimersen for a mean 139.6 weeks. Treatment-emergent adverse events (TEAEs) occurred in all casimersen- and placebo-treated participants and were mostly mild (over 91.4%) and unrelated to casimersen or its dose. There were no deaths, dose reductions, abnormalities in laboratory parameters or vital signs, or casimersen-related serious AEs. Casimersen plasma concentration increased with dose and declined similarly for all dose levels over 24 hours postinfusion. All pharmacokinetic parameters were similar at weeks 7 and 60. DISCUSSION: Casimersen was well tolerated in participants with DMD amenable to exon 45 skipping. Most TEAEs were mild, nonserious, and unrelated to casimersen. Plasma exposure was dose proportional with no suggestion of plasma accumulation. These results support further studies of casimersen in this population.

Bone regeneration in ceramic scaffolds with variable concentrations of PDRN and rhBMP-2.

This study evaluated the bone regeneration capacity and mechanical properties of block-type hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds in response to different concentrations of polydeoxyribonucleotide (PDRN) and recombinant human bone morphogenic protein 2 (rhBMP-2). Thirty-two male white rabbits were used as a model of calvarial bone defect and classified into eight groups according to type and concentration of growth factor administered, viz., control group (only HA/TCP scaffold), scaffold + PDRN (0.1, 1, 5, and 10 mg/mL each) and scaffold + rhBMP-2 (0.01, 0.05, and 0.1 mg/mL each). The specimens were evaluated using histomorphometric and radiological analyses. Histomorphometric analyses indicated that the administration of PDRN did not increase bone formation. However, significant increases in bone formation were observed with the administration of rhBMP-2 at 0.05 and 0.10 mg/mL on week 8 compared to the control (p < 0.05). Radiological analyses revealed a significant increase in bone formation at week 8 with the administration of PDRN at 5 mg/mL and 10 mg/mL, and rhBMP-2 at 0.05 or 0.10 mg/mL compared to the control (p < 0.05). Our findings show that block-type HA/TCP scaffolds possess sufficient mechanical strength and bone regeneration capacity when used with optimal concentrations of growth factors.

Population pharmacokinetics-based recommendations for a single delayed or missed dose of nusinersen.

Nusinersen is an antisense oligonucleotide approved for the treatment of spinal muscular atrophy. The drug is given intrathecally at 12 mg, beginning with 3 loading doses at 2-week intervals, a fourth loading dose 30 days thereafter, and maintenance doses at 4-month intervals. This population pharmacokinetic model was developed to clarify how to maintain targeted nusinersen exposure after an unforeseen one-time delay or missed dose. Simulations demonstrated that the impact of a one-time delay in dosing or a missed dose on median cerebrospinal fluid exposures depended on duration of interruption and the regimen phase in which it occurred. Delays in loading doses delayed reaching the peak trough concentration by approximately the duration of the interruption. Resumption of the regimen as soon as possible resulted in achieving steady state trough concentration upon completion of the loading phase. A short delay (30-90 days) during the maintenance phase led to prolonged lower median cerebrospinal fluid concentration if all subsequent doses were shifted by the same 4-month interval. However, administration of the delayed dose, followed by the subsequent dose as originally scheduled, rapidly restored trough concentration. If a dose must be delayed, patients should return to the original dosing schedule as soon as possible.

Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides.

The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.

Viltolarsen in Japanese Duchenne muscular dystrophy patients: A phase 1/2 study.

OBJECTIVE: The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne muscular dystrophy (DMD). We investigated viltolarsen's ability to induce dystrophin expression and examined its safety in DMD patients. METHODS: In this open-label, multicenter, parallel-group, phase 1/2, exploratory study, 16 ambulant and nonambulant males aged 5-12 years with DMD received viltolarsen 40 or 80 mg/kg/week via intravenous infusion for 24 weeks. Primary endpoints were dystrophin expression and exon 53 skipping levels. RESULTS: In western blot analysis, mean changes in dystrophin expression (% normal) from baseline to Weeks 12 and 24 were - 1.21 (P = 0.5136) and 1.46 (P = 0.1636), respectively, in the 40 mg/kg group, and 0.76 (P = 0.2367) and 4.81 (P = 0.0536), respectively, in the 80 mg/kg group. The increase in mean dystrophin level at Weeks 12 and 24 was significant in the 80 mg/kg group (2.78%; P = 0.0364). Patients receiving 80 mg/kg showed a higher mean exon 53 skipping level (42.4%) than those receiving 40 mg/kg (21.8%). All adverse events were judged to be mild or moderate in intensity and none led to study discontinuation. INTERPRETATION: Treatment with viltolarsen 40 or 80 mg/kg elicited an increasing trend in dystrophin expression and exon 53 skipping levels, and was safe and well tolerated. The decline in motor function appeared less marked in patients with higher dystrophin levels; this may warrant further investigation. This study supports the potential clinical benefit of viltolarsen.

Inotersen for the Treatment of Hereditary Transthyretin Amyloidosis.

Hereditary transthyretin amyloidosis (hATTR) is a rare autosomal dominant condition in which mutations in the transthyretin gene cause amyloid fibrils to develop and deposit into tissues, affecting primarily the nerves and heart causing polyneuropathy and cardiomyopathy respectively. Standard treatment has been liver transplants to try and eliminate the mutated transthyretin products as the liver is the main source of transthyretin production. A new drug named inotersen (brand name Tagsedi), also known as IONIS-TTRRX, has been approved by the United States Food and Drug Agency, Health Canada, and European Commission in 2018, and introduced to the market for patients in stage 1 and stage 2 hATTR polyneuropathy. Inotersen is a second-generation antisense oligonucleotide with 2'-O-methoxyethyl modification designed to bind to the 3' untranslated region of the transthyretin mRNA in the nucleus of the liver cells. By doing so, it prevents the production of the mutant and wild-type forms of transthyretin, impeding the progression of the disease. In this article, the mechanism of action and safety profile of inotersen will be discussed along with some future directions following its approval.

Calcium-Mediated In Vitro Transfection Technique of Oligonucleotides with Broad Chemical Modification Compatibility.

Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca2+-enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.

Phase 1-2 Trial of Antisense Oligonucleotide Tofersen for SOD1 ALS.

BACKGROUND: Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 (SOD1) messenger RNA to reduce SOD1 protein synthesis. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis (ALS) due to SOD1 mutations. METHODS: We conducted a phase 1-2 ascending-dose trial evaluating tofersen in adults with ALS due to SOD1 mutations. In each dose cohort (20, 40, 60, or 100 mg), participants were randomly assigned in a 3:1 ratio to receive five doses of tofersen or placebo, administered intrathecally for 12 weeks. The primary outcomes were safety and pharmacokinetics. The secondary outcome was the change from baseline in the cerebrospinal fluid (CSF) SOD1 concentration at day 85. Clinical function and vital capacity were measured. RESULTS: A total of 50 participants underwent randomization and were included in the analyses; 48 participants received all five planned doses. Lumbar puncture-related adverse events were observed in most participants. Elevations in CSF white-cell count and protein were reported as adverse events in 4 and 5 participants, respectively, who received tofersen. Among participants who received tofersen, one died from pulmonary embolus on day 137, and one from respiratory failure on day 152; one participant in the placebo group died from respiratory failure on day 52. The difference at day 85 in the change from baseline in the CSF SOD1 concentration between the tofersen groups and the placebo group was 2 percentage points (95% confidence interval [CI], -18 to 27) for the 20-mg dose, -25 percentage points (95% CI, -40 to -5) for the 40-mg dose, -19 percentage points (95% CI, -35 to 2) for the 60-mg dose, and -33 percentage points (95% CI, -47 to -16) for the 100-mg dose. CONCLUSIONS: In adults with ALS due to SOD1 mutations, CSF SOD1 concentrations decreased at the highest concentration of tofersen administered intrathecally over a period of 12 weeks. CSF pleocytosis occurred in some participants receiving tofersen. Lumbar puncture-related adverse events were observed in most participants. (Funded by Biogen; ClinicalTrials.gov number, NCT02623699; EudraCT number, 2015-004098-33.).

Brain Targeted Gold Liposomes Improve RNAi Delivery for Glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). METHODS: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. RESULTS: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. DISCUSSION: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders.

Thiol-responsive pro-fluorophore labeling: Synthesis of a pro-fluorescent labeled oligonucleotide for monitoring cellular uptake.

Pro-fluorescent labeled oligonucleotides are potential alternative tools to classical fluorescently labeled oligonucleotides for monitoring cellular uptake. Here, we report the design and synthesis of a thiol-responsive pro-fluorophore labeled oligonucleotide, and its fluorescence responsivity to glutathione in the test tube and live cells.

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Population Pharmacokinetic-Pharmacodynamic Modeling of Inotersen, an Antisense Oligonucleotide for Treatment of Patients with Hereditary Transthyretin Amyloidosis.

A population pharmacokinetic (PK) and pharmacodynamic (PD) model was developed for inotersen to evaluate exposure-response relationships and to optimize therapeutic dosing regimen in patients with hereditary transthyretin (TTR) amyloidosis polyneuropathy (hATTR-PN). Inotersen PK and TTR level (PD) data were composed of one Phase 1 study in healthy subjects, one Phase 2/3 study in hATTR patients, and its one open-label extension study. Effects of intrinsic and extrinsic factors (covariates) on PK and PK/PD of inotersen were evaluated using a full model approach. Inotersen PK was characterized by a two-compartment model with elimination from the central compartment. The population PK analysis identified disease status and lean body mass (LBM) as significant covariates for inotersen PK. Nonetheless, the contribution of disease status and LBM on PK was small, as the difference in clearance (CL/F) was 11.1% between healthy subjects and patients with hATTR-PN and 38% between the lowest and highest LBM quartiles of the patient population. Age, race, sex, baseline renal function estimated glomerular filtration rate, and hepatic function markers (baseline albumin, bilirubin, and alanine aminotransferase values) were not statistically significant covariates affecting inotersen PK. An inhibitory effect indirect-response model (inhibition of TTR production) was used to describe the drug effect on TTR-time profiles, with baseline TTR included as a covariate. The overall population Imax and IC50, together with 95% confidence interval, was estimated to be 0.913 (0.899-0.925) and 9.07 (8.08-10.1) ng/mL, respectively. V30M mutation showed no effect on the estimated IC50 value for hATTR patients. The final population PK and PK/PD model was used to simulate four different treatment regimens. The population PK/PD model developed well described the PK and PD of inotersen in patients with hATTR-PN and has been used for label recommendation and trial simulations.

Mucoadhesive-to-penetrating controllable peptosomes-in-microspheres co-loaded with anti-miR-31 oligonucleotide and Curcumin for targeted colorectal cancer therapy.

Background: Accumulating evidences indicate that nanomedicines greatly decrease the side effects and enhance the efficacy of colorectal cancer (CRC) treatment. In particular, the use of rectal delivery of nanomedicines, with advantages such as fast therapeutic effects and a diminishing hepatic first-pass effect, is currently emerging. Method: We established a CRC targeted delivery system, in which α-lactalbumin peptosomes (PSs) co-loaded with a microRNA (miR)-31 inhibitor (miR-31i) and curcumin (Cur) were encapsuslated in thiolated TEMPO oxidized Konjac glucomannan (sOKGM) microspheres, referred as sOKGM-PS-miR-31i/Cur. The CRC targeting capability, drug release profiles, mucoadhesive-to-penetrating properties and therapeutic efficacy of sOKGM-PS-miR-31i/Cur delivery system were evaluated in colorectal cancer cells and azoxymethane-dextran sodium (AOM-DSS) induced tumor models. Results: sOKGM-PS-miR-31i/Cur delivery system were stable in the harsh gastrointestinal environment after rectal or oral administration; and were also mucoadhesive due to disulfide bond interactions with the colonic mucus layer, resulting in an enhanced drug retention and local bioavailability in the colon. Concomitantly, the released PS-miR-31i/Cur PSs from the microsphere was mucus-penetrating, efficiently passing through the colonic mucus layer, and allowed Cur and miR-31i specifically target to colon tumor cells with the guide of CD133 targeting peptides. Consequently, rectal delivery of sOKGM-PS-miR-31i/Cur microspheres suppressed tumor growth in an azoxymethane-dextran sodium sulfate (AOM-DSS)-induced tumor model. Conclusion: sOKGM-PS-miR-31i/Cur microspheres are effective rectal delivery system with combined advantages of mucoadhesive and mucus-penetrating properties, representing a potent and viable therapeutic approach for CRC.

Synthesis and applications of theranostic oligonucleotides carrying multiple fluorine atoms.

The use of various oligonucleotide (ON) syntheses and post-synthetic strategies for targeted chemical modification enables improving their efficacy as potent modulators of gene expression levels in eukaryotic cells. However, the search still continues for new approaches designed for increasing internalization, lysosomal escape, and tissue specific delivery of ON. In this review we emphasized all aspects related to the synthesis and properties of ON derivatives carrying multifluorinated (MF) groups. These MF groups have unique physico-chemical properties because of their simultaneous hydrophobicity and lipophobicity. Such unusual combination of properties results in the overall modification of ON mode of interaction with the cells and making multi-fluorination highly relevant to the goal of improving potency of ON as components of new therapies. The accumulated evidence so far is pointing to high potential of ON probes, RNAi components and ON imaging beacons carrying single or multiple MF groups for improving the stability, specificity of interaction with biological targets and delivery of ONs in vitro and potentially in vivo.